Structural studies of protein-protein interactions of the IgE network

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

The incidence of allergic disease in the UK is already high and is increasing. There is an urgent need for new therapies, and the long-term aim of this project is to further the development of new therapeutic compounds. Antibodies of a type known as immunoglobulin E (IgE) are crucially involved in the allergic response, and individuals susceptible to allergies have high levels of IgE in the blood. The IgE antibodies are the link between the allergens that enter the airways (such as grass pollen and house dust mites), the gut (food allergens) or the skin (contact allergies), and the body?s cells that trigger the allergic reaction. These are called mast cells, and the IgE antibodies bind to them through molecules on the cell surface known as ?receptors?. If we can block the binding of the IgE antibodies to the receptors, then all of these manifestations of allergic disease would be prevented.

In order to design compounds that will block this step in the allergic reaction, we need to know the detailed molecular structures of IgE and the receptor. We use both X-ray and magnetic resonance imaging techniques applied to individual molecules, to work out their atomic structures. Using these same techniques, we can also study the way in which the molecules bind to each other. In this way we shall build up an understanding at the molecular level of how the allergic response works. We also plan to study the molecular interactions that control the levels of IgE production in the body, and we have already set up experimental systems using nasal and lung biopsy material from patients with allergic rhinitis and asthma, to test the effects of potential inhibitors.

This programme of work provides the starting point for the design of compounds to inhibit these interactions. Such compounds are potential therapeutic agents for the treatment of allergic disorders.

Technical Summary

Allergic disease now affects one person in six in the UK, and the incidence of certain conditions such as childhood asthma is increasing. Interactions between immunoglobulin E (IgE) and its receptors are crucially involved at various stages of the allergic response, including the presentation of allergens to the immune system, the regulation of IgE synthesis and perhaps also the switch to IgE production by B cells, and the triggering of mast cells and basophils in the immediate hypersensitivity reaction. We have embarked upon a programme of structural studies of these proteins and their complexes - IgE and IgE Fc, FceRI (the high affinity mast cell receptor), FceRII/CD23 (the low affinity B cell receptor), CR2/CD21 (the counter-receptor for CD23 on B cells) - by X-ray crystallography, NMR and other biophysical techniques. Understanding the molecular details of these interactions, all of which are potential points of therapeutic intervention, is essential for the design and/or screening for blocking agents. We have recently determined the crystal structure of the complete IgE Fc [1], and shown by NMR that the Ce2 domains of IgE interact with FceRI [2]; studies of CD23 by both X-ray crystallography and NMR are in progress. Future structural studies of their complexes require the production of large amounts of many different soluble versions of these various proteins. Site-specific mutant versions of these proteins will also be produced for NMR and BIAcore experiments to investigate the kinetics of the interactions and the role of key residues. These protein fragments are themselves potential inhibitors, and will be tested in functional assays for IgE synthesis and allergen presentation. These assays have been developed through collaborations within the Co-operative Group: for IgE synthesis, between HJG and Prof. Stephen Durham and Dr. Chris Corrigan, and for allergen presentation, between HJG and Dr. Catherine Hawrylowicz (the latter two in Prof. Tak Lee?s Department).

This application is for technical assistance to support these structural and functional studies, through the expression of the various recombinant proteins in different systems (mammalian, yeast, bacterial), their purification, characterisation and crystallisation. This support will underpin the collaboration between the three applicants, together with four other members of the Co-operative Group. The scientific aims of this project are central to the long-term goals of the Group, namely the development of therapeutic agents for intervention in allergic disease.

Publications

10 25 50
 
Description Asthma UK Consultation on Basic Asthma Research Strategy
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description Asthma UK Consultation on Basic Asthma Research Strategy
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description Asthma UK Project Grant
Amount £143,000 (GBP)
Organisation Asthma UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2006 
End 01/2008
 
Description Asthma UK Project Grant
Amount £200,000 (GBP)
Organisation Asthma UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2009 
End 10/2011
 
Description Biomedical Research Centre, Programme Grant
Amount £500,000 (GBP)
Organisation National Institute for Health Research 
Department NIHR Comprehensive Biomedical Research Centre, Guy's and St Thomas
Sector Public
Country United Kingdom
Start 01/2009 
End 01/2011
 
Description CRUK Project Grant
Amount £205,428 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2009 
End 01/2011
 
Description Guy's and St. Thomas' Charity/Novartis Clinical Trial
Amount £300,000 (GBP)
Organisation Novartis 
Sector Private
Country Global
Start 01/2009 
End 01/2011
 
Description Guy's and St. Thomas' Charity/Novartis Clinical Trial
Amount £300,000 (GBP)
Organisation Guy’s & St Thomas’ Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2009 
End 01/2011
 
Description Guy's and St. Thomas's Trust Charity, Project Grant
Amount £261,554 (GBP)
Organisation Guy’s & St Thomas’ Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2009 
End 01/2011
 
Description Heptagon Fund
Amount £26,000 (GBP)
Organisation Heptagon Fund 
Sector Private
Country United Kingdom
Start 10/2006 
End 09/2007
 
Description MRC Programme Grant
Amount £2,323,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2006 
End 09/2011
 
Description MRC/EPSRC Project Grant
Amount £670,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2007 
End 01/2011
 
Description Translational Research Award
Amount £98,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 08/2008 
End 04/2009
 
Description Wellcome Trust Programme Grant
Amount £817,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2006 
End 01/2011
 
Title Cell lines producing recombinant IgE 
Description Improved cell lines producing IgE antibodies 
Type Of Material Cell line 
Year Produced 2007 
Provided To Others? Yes  
Impact Ongoing trials of IgE for cancer immunotherapy 
 
Title Fluorescently labelled IgE biosensors 
Description Fluorescently labelled versions of IgE-Fc that are sensitive to conformational changes in the IgE molecule. Valuable for screening for small molecules that influence the structure of IgE and that might therefore be leads for IgE inhibitors. 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact Currently under investigation within our group; publications in preparation; potential IgE small molecule inhibitors. 
 
Title High resolution IgE-Fc structure 
Description Crystal structure of IgE-Fc determined at high resolution (1.9 Angstroms); valuable for anti-IgE drug discovery; PDB code 2WQR 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact This structure has enabled in silico and rational design of anti-IgE inhibitors, which are currently being evaluated. 
URL http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=2WQR
 
Title IgE-Fc crystal structure 
Description Crystal structure of IgE-Fc. Coordinates deposited in the Protein Data Bank code 2wqr. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact This structure of IgE-Fc revealed the molecule to be acutely bent, and this led to understanding that allosteric approaches to inhibiting IgE activity might be possible. 
URL http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=2WQR
 
Title Mouse model for allergy and cancer immunotherapy 
Description Transgenic mouse model for intervention in allergic disease and for immunotherpy for cancer. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact On-going development programme for small molecule inhibitors of IgE/receptor interactions in allergy, and on-going trials of IgE in cancer immunotherapy. 
 
Title Structure of IgE-Fc and receptor complex 
Description X-ray crystal structure at high resolution of IgE-Fc (PDB code 2wqr), and also the structure of the complex with receptor. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact The structural data reveal conformational changes that can be targetted in the design of an allosteric inhibitor of the IgE/receptor interaction. A drug discovery programme is in progress, both in-house and as an industrial collaboration. 
URL http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=2WQR
 
Description Dr. David Dombrowicz 
Organisation Pasteur Institute, Paris
Country France 
Sector Academic/University 
PI Contribution Material and labour and expertise in IgE, CD23 and FceRI.
Collaborator Contribution Transgenic mouse model of allergy and cancer.
Impact Publications; proof of concept for inhibition of IgE/FceRI for treatment of allergy, and also for monoclonal antibodies in immunotherapy of cancer; development of an animal models.
 
Description Novartis 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution Expertise in IgE and clinical trial facilities and patients.
Collaborator Contribution Provision of anti-IgE and manpower to conduct the experiments
Impact A clinical trial has been initiated as a result of the collaboration; additional funding was obtained from other sources; understanding of role of IgE in non-atopic asthma
Start Year 2009
 
Description Professor Dan Conrad 
Organisation Virginia Commonwealth University
Country United States 
Sector Academic/University 
PI Contribution Provision materials, carrying out experimental work, biophysical characterisation etc.
Collaborator Contribution Expertise in functional assays for role of CD23 in IgE synthesis
Impact Publications; undertanding of the role of CD23 in IgE regulation and production in B cells.
 
Description Professor Erika Jensen-Jarolim 
Organisation Medical University of Vienna
Country Austria 
Sector Academic/University 
PI Contribution Expertise in IgE function; recombinant DNA technology; materials and facilities for the functional studies, all of which were performed at King's.
Collaborator Contribution Two students from the Vienna lab worked at King's on functional studies of chimeric monoclonal IgEs against ovarian cancer.
Impact Several publications; further funding from BRC and CRUK; a clinical trial of a monoclonal IgE (in progress); studentships from BRC.
Start Year 2007
 
Description Professor Michael Holers 
Organisation University of Colorado Denver
Country United States 
Sector Academic/University 
PI Contribution Provision of CD23 and other reagents
Collaborator Contribution Provision of materals.
Impact Publications (see list); understanding the role of CD23 and complement receptor (CD21) interactions in autoimmune disease, and allergy.
 
Description Professor Toshi Kawakami 
Organisation Los Angeles Institute of Allergy and Clinical Immunology
Country United States 
Sector Academic/University 
PI Contribution Provision of cDNA of potentially cytokinergic IgE for analysis.
Collaborator Contribution Expertise in analysis of cytokinergic IgE; sequence data.
Impact Demonstration that human IgE can exert cytokinergic activity; this has profound implications for the pathogenesis of allergic disease and asthma in particular.
Start Year 2007
 
Title Anti-IgE antibodies 
Description Antibody-derived biologic that inhibits binding of IgE to its receptors on effector cells. 
IP Reference WO2017211928 
Protection Patent application published
Year Protection Granted 2017
Licensed No
Impact Understanding of the mechanism of action of anti-IgE antibodies in general and the possibilities for allosteric inhibition.
 
Description Asthma UK Website for World Asthma Day 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Report of our asthma research to develop a novel therapy was highlighted on the website and in publicity material distributed in association with World Asthma Day, 2009.

Raised profile of the need for more research funding and clinical provision for asthma.
Year(s) Of Engagement Activity 2009,2011
 
Description BBC Radio 4 Programme 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Contributed to a BBC Radio Programme on Asthma provision and asthma research: Our Lives in Our Hands - Asthma, Living on a Knife Edge.

Raised profile of the life-threatening aspect of asthma and allergic disease, and of the widespread nature of asthma.
Year(s) Of Engagement Activity 2006
 
Description Interview about Rosalind Franklin (Mars Rover naming; 07/02/19) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interview with BBC Radio Wales and contribution to BBC TV (World News) explaining the role of Rosalind Franklin in the discovery of the structure of DNA, following the naming of the Mars Rover that will search for evidence of life on Mars.
Year(s) Of Engagement Activity 2019
 
Description Interview for "New Scientist" article on the Allergy Epidemic (22/05/18) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Contribution to widely-read article on the "Allergy Epidemic".
Year(s) Of Engagement Activity 2018
 
Description Interview for "Technology Network" (22/06/18) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Interview and article about X-ray crystallography and structural biology.
Year(s) Of Engagement Activity 2018
 
Description Newspaper and radio interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Articles and interviews concerning the trials of IgE in the immunotherapy of cancer

Raised awareness of this approach with the public and assisted in gaining funding.
Year(s) Of Engagement Activity 2007,2008,2009,2013
 
Description Newspaper and radio interviews 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Articles and interviews concerning the trials of IgE in the immunotherapy of cancer

Raised awareness of this approach with the public and assisted in gaining funding.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Pattern in African fabric design (12/02/19) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Contribution to a "King's Together" interdisciplinary project to explore pattern in African textile design; my contribution is to classify pattern and symmetry.
Year(s) Of Engagement Activity 2019
 
Description Presentations at Asthma UK events 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presented my research on IgE and the development of novel therapeutics to fundraisers, supporters and patients at events organised by Asthma UK.

Raises profile of asthma research nationally. Assisted the charity in attracting new donors and raising funds for asthma research.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Production of "Photograph 51" (Cambridge) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Talk and presentation of artefacts in the King's College London Archive to the cast and production team of a new production of "Photograph 51", a play by Anna Ziegler, in Cambridge in April 2019. The cast reported that they were inspired by their new-found understanding of X-ray diffraction, DNA and the scientists involved in the discovery of its structure.
Year(s) Of Engagement Activity 2019
 
Description Public talk (22/03/18 at Guy's Campus of KCL) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk about symmetry and molecular structure, in particular handedness (chirality) and its discovery. This was followed by a hands-on event, generating a quasi-periodic tiling pattern. Comments afterwards from many showed that a whole new way of looking at molecular structure had been opened up for them.
Year(s) Of Engagement Activity 2018
 
Description School visit (Greycoat School 21/11/18) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk about DNA and role of Rosalind Franklin in the discovery of its structure.
Year(s) Of Engagement Activity 2018
 
Description Seminar on novel asthma/allergy therapy development 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presented at several activities organised by Asthma UK aimed at their fundraisers, other supporters and of course including patients.

Raied profiule for our work, and the role of structural biology in the drug discovery process. Enhanced the ability of the charity to engage new donors and raise funds for asthma research.
Year(s) Of Engagement Activity 2006,2007,2008,2009
 
Description Seminars on development of IgE inhibitors 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact Various talks to companies, under confidentiality agreements (UCB Celltech, UK; AstraZeneca, Sweden; Pfizer, UK & USA)

We successfully negotiated an agreement with a company for collaborative development of IgE inhibitors.
Year(s) Of Engagement Activity 2007,2008,2009
 
Description Seminars on development of IgE inhibitors for anti-allergy therapy 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact Discussions under confidentiality agreements, with various companies, recently UCB-Celltech (UK), AstraZeneca (Sweden) and Pfizer (UK and USA).

Negotiations concluded with one UK-based company; unable to provide further details at present.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Sixth Form talks on Asthma, Allergy and Drug Design 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Talks to Sixth Forms, most recently at Magdalen College School, Oxford, and St. Paul's School, London.

Very positive feedback from school staff about pupils' future subject choices at university and career plans. Led to collaborative project with St. Paul's School sixth form; students spend day in lab at King's.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Sixth Form talks on Asthma, Allergy and Drug Design 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Most recently to St. Paul's School, London.

Positive feedback from teachers about pupils' university choices and career plans. This talk led to initiation of a project to bring sixth formers into King's to carry out an experiemtnal project involving molecular and structural biology. First run in 2009, the scheme was a great success and will be repeated annually.
Year(s) Of Engagement Activity 2008,2009,2010,2014
 
Description Talk and hands-on tiling event (30/05/18) Bush House Arcade 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Hands-on event to generate a novel quasi-periodic tiling pattern involving tiles with handedness (chirality). Brief talk also delivered twice during the day. Considerable interest generated and discussions promoted about the nature of handedness, crystals and molecular structure, periodicity and quasi-symmetry.
Year(s) Of Engagement Activity 2018
 
Description Talk to Aldwych Quarter Project (25/07/18) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk about proposals for tiling patterns as part of the Aldwych Quarter development by Westminster City Council.
Year(s) Of Engagement Activity 2018
 
Description Talk to King's Designers (28/06/18) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk about symmetry, handedness and pattern. Many in the audience reported that they now viewed pattern and symmetry in an entirely new way.
Year(s) Of Engagement Activity 2018
 
Description Telephone interviews with journalists 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Telephone interviews with journalists about our IgE work and development of new drugs. Press releases accompanied recent publication in J. Biol. Chem. Reports in the Times, Daily Telegraph, Daily Mail, London Metro, NHS Web pages.

Raises profile of our work nationally, and of the need for research into allergy and asthma generally.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Telephone interviews with newspapers 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Various interviews leading to pieices in newspapers: Times, Daily Telegraph, Sunday Times, Daily Mail, London Metro, NHS Web pages. (Earlier years: breakfast TV appearance and Radio 4 programme "Leading Edge")

Raised national public profile of allergy research generally.
Year(s) Of Engagement Activity 2007,2008
 
Description Video about Rosalind Franklin (06/12/18) 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Video to explain role of Rosalind Franklin in the discovery of the structure of DNA, promoted by King's College London.
Year(s) Of Engagement Activity 2018